Method for treating circadian rhythm disruptions

ABSTRACT

A neuropeptide Y Y5 antagonist is useful, alone or in conjunction with other agents, for altering circadian rhythmicity and alleviating circadian rhythm disorders and for enhancing and improving the quality of sleep. The present invention further provides for the use of a medicament for carrying out these methods.

BACKGROUND OF THE INVENTION

Circadian rhythms are exhibited by all eukaryotic plants and animals,including man. Biological rhythms are periodic fluctuations inbiological processes over time, including circadian as well as seasonalvariations. Circadian, or approximately 24-hour, rhythms include theproduction of biological molecules such as hormones, the regulation ofbody temperature, and behavior such as wakefulness, alertness, sleep andperiods of activity. Circadian rhythms are endogenous, self-sustainedoscillations over 24-hour periods found in organisms ranging fromprokaryotes to humans (J S Takahashi, et al. Science, 217, 1104-1111(1982)).

In nature, circadian rhythms are closely tied to environmental cues thatimpose a 24-hour pattern on many of these fluctuations. The regulationof circadian rhythms by signals from the environment involves“entrainment” of the circadian rhythm. The environmental signals whichaffect entrainment of the circadian rhythm are termed “zeitgebers”, anexample of which is the light-dark cycle.

The control of many circadian rhythms in mammals is mediated by theportion of the brain called the suprachiasmatic nuclei (SCN). In humansas well as other mammals, the circadian clock, which controls allendogenous circadian rhythms, is located in the SCN of the hypothalamus.Activity, alertness, core body temperature, and many hormones all haveendogenous circadian rhythms controlled by the SCN. The SCN is theprimary pacemaker for circadian rhythms in mammals. Circadian rhythmsare primarily entrained by the environmental light-dark cycle. One ofthe most important and reproducible characteristics of a circadian clockis that it can respond to exogenous light/dark signals. The circadianclock is composed of three parts: light-input pathways, a clock, andeffector (“output”) pathways. Light signals are conveyed by the retinato the SCN, and the pineal gland produces melatonin(N-acetyl-5-methoxytryptamine), which is regulated by the SCN.Information regarding light is conveyed from the retina to the SCN viathe direct retinohypothalamic tract (RHT), as well as indirectly via thelateral geniculate nucleus (LGN) (D C Klein et al., (1991)Suprachiasmatic nucleus: the mind's clock. New York: Oxford UniversityPress).

It has been suggested in the art that excitatory amino acids areinvolved in the transduction of information regarding the light-darkcycle to the SCN. Acetylcholine, neuropeptide Y, GABA, 5HT₁ receptorfunctioning, glutamate, and substance P receptor may play a role in theentrainment and/or generation of circadian rhythms in mammals. Theoscillator in the SCN can be reset by photic input, which is mediated byglutamatergic afferents originating in the retina. Glutamate can mimicthe effects of light on the mammalian circadian clock in vitro. BothNMDA glutamate receptors and non-NMDA receptors can mediate the effectsof light on the circadian clock (J M Ding, et al. Science, 266,1713-1717 (1994); S. Shibata, et al. Am. J. Physiol., 267, R360-R364(1994)). Application of NMDA in vitro can phase-shift electricalactivity rhythms in hypothalamic brain slices containing the SCN.Activation of NMDA receptors is believed to be an important step in thetransmission of photic information to the SCN (E M Mintz, et al., J.Neurosci., 19, 5124-30 (1999).

Neuropeptide Y (NPY) is a 36 amino acid peptide that is a member of thepancreatic polypeptide family, which also includes pancreaticpolypeptide (PP) and peptide YY (PYY). NPY is located throughout thecentral and peripheral nervous systems and affects a diverse range ofbiological functions, including central endocrine secretion, vascularand smooth muscle activity, appetite, memory, anxiety, blood pressureregulation and reproduction. See, e.g., Karla, et al., Phys. & Behavior50, 5 (1991).

NPY receptors are members of the G protein-coupled receptor superfamily.At present, NPY is known to bind to at least five receptors: Y1, Y2, Y3,Y4 and. Y5. Both Y1 and Y5 NPY receptors are expressed in the SCN (P JLarsen and P Kristensen, Mol. Brain Res. 60, 69-76 (1998). It is thoughtthat a Y5 antagonist can prevent the effect of NPY on NMDA-induced phaseshifts of the SCN circadian neural activity rhythm. The Y5 receptormediates the blocking effect of NPY on NMDA-induced phase shifts.

The SCN and the circadian clock control the phases and rhythms of anumber of hormonal rhythms in humans. One of the most well-characterizedSCN outputs is to the pineal body, via a circuitous route from thehypothalamus to the spinal cord and then back to the pineal. The humanpineal gland secretes melatonin in a circadian fashion, such that theplasma concentrations observed during the night are ten to forty timeshigher than those observed during the day. This plasma melatonin rhythmis a true circadian rhythm, and therefore not dependent upon theexogenous light-dark cycle, as it persists in blinded animals and blindhumans. However, light is able to influence the endogenous melatoninrhythm. Light exposure during the night, when plasma melatoninconcentrations are high, is able to rapidly suppress plasma melatonin tonear daytime levels in a dose-dependent manner (C A Czeisler, et al. N.Eng. J. Med., 332, 6-11 (1995); McIntyre I M, et al. J Pineal Res, 6,149-56 (1989); D B Boivin, et al. Nature, 379, 540-2 (1996)). Thesuppressive effects of light on plasma melatonin concentrations arebelieved to be mediated through the retina-SCN-pineal neural pathway (RY Moore, et al. Science, 210, 1267-9 (1980)). It is believed that a NPYY5 agonist can block the NMDA-induced phase shift (delay) of SCNcircadian neuronal activity. A NPY Y5 receptor antagonist mightfacilitate “resetting” of the clock by inducing sleep at clock-relevanttimes, much like melatonin. A NPY Y5 receptor antagonist is expected toalter photic entrainment of the circadian clock in vivo.

Circadian rhythms are also an important modulator of sleep. Althoughsleep is necessary for survival, its precise homeostatic contribution isunknown. Sleep is not a uniform state, but rather involves severalstages characterized by changes in the individual's EEG. A non rapid eyemovement (NREM) type (75 to 80% of total sleep time) ranges in depththrough stages 1 to 4 (deepest level). Stage 1 sleep is drowsiness, inwhich the EEG displays a lower voltage, more mixed frequencies anddeterioration of alpha rhythm relative to the EEG when the individual isawake. In stage 2, background activity similar to that of stage 1 isexperienced, with bursts of slightly higher frequency “sleep spindles”and sporadic higher amplitude slow wave complexes. The third and fourthstages of sleep display increasing high amplitude slow wave activity.The separate sleep stage in which the individual undergoes rapid eyemovement (REM) occupies the remainder of the sleep time and occurs 5 to6 times during a normal nights sleep. REM sleep is characterized by alower voltage, higher frequency EEG and other characteristics similar tothose which occur when the individual is awake, whereas the other foursleep stages are categorized as NREM sleep.

Individuals vary widely in their requirements for sleep, which isinfluenced by a number of factors including their current emotionalstate. The natural aging process is associated with changes in a varietyof circadian and diurnal rhythms. Age-related changes in the timing andstructure of sleep are surprisingly common problems for older people,and are often associated with significant morbidity. With advancing age,the total amount of sleep tends to shorten. Stage 4 can decrease ordisappear and sleep may become more fragmented and interrupted.Evaluation of sleep patterns in elderly people shows that the timing ofsleep is also phase advanced, especially in women. This tendency to goto sleep and wake up earlier is very frustrating to older people whofeel that they are out of step with the rest of the world. In addition,the quality of sleep in the elderly is diminished with a markedreduction in slow wave sleep, a reduction in the deep stages of sleep(especially stage 4), fragmentation of REM sleep and more frequentawakenings. Similarly, non-elderly people may exhibit disturbances inthe normal sleep process. These changes in the structure of sleep havebeen correlated to more frequent napping, decreased daytime alertnessand declining intellectual function and cognitive ability. Deprivationof REM sleep has been suggested to interfere with the memoryconsolidation involved in learning skills through repetitive activity,and slow wave sleep has been implicated as being important inconsolidation of events into long term memory. Likewise, decreases inthe length of REM stages of sleep may be associated with a decrease incognitive function and learning, especially diminished retention ofmemory. Depression and insomnia may involve a disruption of normalcircadian rhythmicity.

Sleep disorders generally involve disturbances of sleep, includingcircadian rhythm disturbances, that affect a subject's ability to falland/or stay asleep, and involve sleeping too little, too much orresulting in abnormal behavior associated with sleep.

Numerous compounds are employed in the art to facilitate normal sleepand to treat sleep disorders and sleep disturbances, including e.g.,sedatives, hypnotics, anxiolytics, antipsychotics, antianxiety agents,minor tranquilizers, melatonin receptor agonists and antagonists,melatonergic agents, benzodiazepines, barbiturates, 5HT-2 antagonists,and the like. Similarly, physical methods have been employed to treatpatients with sleep disorders such as the use of light therapy or theapplication of modulated electrical signals to selected nerves or nervebundles.

Nevertheless, the known therapeutic regimens suffer from numerousproblems, including residual effects in daytime function, impairment ofmemory, potential for addiction, rebound insomnia, “REM rebound” whichmay be associated with increased dream intensity and the occurrence ofnightmares, and the like. Accordingly, a more physiological way toenhance sleep, achieve a chronobiologic (circadian rhythmphase-shifting) effect or alleviate circadian rhythm sleep disorderswould be highly desirable.

SUMMARY OF THE INVENTION

The present invention relates to the use of a NPY Y5 antagonist forachieving a chronobiologic (circadian rhythm phase-shifting) effect andalleviating circadian rhythm disorders in a mammal. The presentinvention is further directed to the use of a NPY Y5 antagonist, forblocking the phase-shifting effects of light in a mammal. Accordingly,the present invention provides a method for achieving a circadian rhythmphase-shifting effect in a mammal comprising the administration of NPYY5 antagonist. The present invention further provides a pharmaceuticalcomposition for achieving a circadian rhythm phase-shifting effect. Thepresent invention further provides a method of manufacture of amedicament useful for achieving a circadian rhythm phase-shiftingeffect, for the treatment or prevention of a circadian rhythm disorder,and for blocking the phase-shifting effects of light.

The present invention further relates to the use of a NPY Y5 antagonistfor enhancing or improving sleep quality, in particular by increasingsleep efficiency and augmenting sleep maintenance, as well as forpreventing and treating sleep disorders and sleep disturbances, in amammal. Accordingly, the present invention provides a method forenhancing or improving sleep quality and increasing sleep efficiency andsleep maintenance in a mammal comprising the administration of NPY Y5antagonist. The present invention further provides a pharmaceuticalcomposition for enhancing or improving sleep quality and increasingsleep efficiency and sleep maintenance. The present invention furtherprovides a method of manufacture of a medicament useful for enhancingand improving the quality of sleep, and for the treatment of sleepdisorders and sleep disturbances.

DESCRIPTION OF THE INVENTION

The present invention is directed to the use of NPY Y5 antagonist, forachieving a chronobiologic (circadian rhythm phase-shifting) effect andalleviating circadian rhythm disorders in a mammal. The presentinvention is further directed to the use of NPY Y5 antagonist, formodulating the phase-shifting effects of light in a mammal.

In an embodiment, the present invention provides a method for the phaseadvance or phase delay in the circadian rhythm of a subject whichcomprises administering to the subject an appropriate amount of a NPY Y5antagonist.

The administration to a subject of an appropriate amount of a NPY Y5antagonist is useful, for example, in the prevention or treatment of thefollowing conditions to achieve chronobiologic (circadian rhythmphase-shifting) effects and/or to alleviate circadian rhythm phasedisorders or disturbances: disorders of the sleep-wake schedule; jetlag; shift work; people who have a maladaption to work and off-workschedules; medical residents, nurses, firemen, policemen or those whoseduties require alertness and wakefulness at evening or nighttime hours,or those deprived of sleep for various periods because of their dutiesor responsibilities; animal workers; athletes who wish to reset theirinternal clock to a more beneficial time; the infantry, or other membersof the armed forces whose duties require extreme levels of alertness andwakefulness, and those who may be sleep deprived in the performance ofthese duties; submariners, or people confined for research, explorationor industrial purposes below the seas; miners, spelunkers, researchersor those confined beneath the Earth; astronauts in orbit around theEarth, on missions in space to the Earth's moon or to the planets or outof the solar system, or in training for such missions; the blind orsight-impaired or those persons whose ability to distinguish differencesin light and dark may be permanently or temporarily impaired;psychiatric patients; those with night eating syndrome, insomniacs; thecomatose, or those who need to be maintained in a state ofunconsciousness for medical, psychiatric or other reasons; residents ofthe far North or Antarctica, or those persons who live in a climate orclimates which possess abnormal amounts of light or darkness; thosesuffering from seasonal affective disorder (SAD), winter depression, orother forms of depression; the aged; Alzheimer's disease patients, orthose suffering from other forms of dementia; patients who requiredosages of medication at appropriate times in the circadian cycles;patients suffering from delayed sleep phase syndrome, advanced sleepphase syndrome, or non-24 hr sleep phase syndrome; and patientssuffering from primary or secondary insomnia or circadian rhythm-relatedinsomnia.

Circadian rhythms affect a variety of physiological parameters:rest-activity, sleep-wake cycles, body temperature, rhythms in hormonelevels, oscillations in general physiology and the like. When theseparameters are out of synchrony with the daily clock, a circadian rhythmimbalance occurs which can affect physiology, performance on a varietyof tasks and one's emotional well being. The present invention isuseful, for example, in the prevention or treatment of conditionsassociated with circadian rhythmicity as well as mental and physicaldisorders associated with travel across time zones and with rotatingshift-work schedules.

In another embodiment, the present invention provides a method for theprevention or treatment of a circadian rhythm disorder in a mammal,including time-zone change (jet-lag) syndrome, shift-work sleepdisorder, delayed sleep-phase syndrome, advanced sleep-phase syndrome,and non-24-hour sleep-wake disorder, which comprises administering tothe mammal an effective amount of a NPY Y5 receptor antagonist.

In another embodiment, the present invention provides a method forshortening the time of re-entrainment (return to normal entrainment ofthe circadian rhythms; synchronized to the environmental light-darkcycle) in a subject following a shift in the sleep-wake cycle whichcomprises administering to the subject an appropriate amount of a NPY Y5antagonist.

In another embodiment, the present invention provides a method foralleviating the effects of jet lag in a traveler, especially a mammal,which comprises administering to the traveler an alertness increasingamount of a NPY Y5 antagonist. The purpose of this embodiment is toassist the body to adjust physiologically to the changes in sleep andfeeding patterns when crossing several time zones.

In another more preferred embodiment, the present invention provides amethod for resetting the internal circadian clock in a subject to matchthe subject's current activity/sleep cycle. For example shift workerschanging from a day to a night shift or vice versa, which comprisesadministering to the subject an appropriate amount of a NPY Y5antagonist.

The present invention is further directed to the use of NPY Y5antagonist, for enhancing or improving sleep quality as well aspreventing and treating sleep disorders and sleep disturbances in amammal. In particular, the present invention provides a method forenhancing or improving sleep quality by increasing sleep efficiency andaugmenting sleep maintenance. In addition, the present inventionprovides a method for preventing and treating sleep disorders and sleepdisturbances in a mammal which comprising the administration of a NPY Y5antagonist. The present invention further provides a pharmaceuticalcomposition for enhancing or improving sleep quality and increasingsleep efficiency and sleep maintenance. The present invention is usefulfor the treatment of sleep disorders, including Disorders of Initiatingand Maintaining Sleep (insomnias) (“DIMS”) which can arise frompsychophysiological causes, as a consequence of psychiatric disorders(particularly related to anxiety), from drugs and alcohol use and abuse(particularly during withdrawal stages), childhood onset DIMS, nocturnalmyoclonus and restless legs and non specific REM disturbances as seen inageing.

The following outcomes in a subject which are provided by the presentinvention may be correlated to enhancement in sleep quality: an increasein the value which is calculated from the time that a subject sleepsdivided by the time that a subject is attempting to sleep; a decrease insleep latency (the time it takes to fall asleep); a decrease in thenumber of awakenings during sleep; a decrease in the time spent awakefollowing the initial onset of sleep; an increase in the total amount ofsleep; an increase the amount and percentage of REM sleep; an increasein the duration and occurrence of REM sleep; a reduction in thefragmentation of REM sleep; an increase in the amount and percentage ofslow-wave (i.e. stage 3 or 4) sleep; an increase in the amount andpercentage of stage 2 sleep; a decrease in the number of awakenings,especially in the early morning; an increase in daytime alertness; andincreased sleep maintenance. Secondary outcomes which may be provided bythe present invention include enhanced cognitive function and increasedmemory retention. A “method for enhancing the quality of sleep” refersto a method that results in outcomes in a subject which may becorrelated to enhancement in sleep quality, including, but not limitedto, the outcomes correlated to enhancement of sleep quality as definedabove.

The present invention is further useful for the prevention and treatmentof sleep disorders and sleep disturbances including sleep problemsassociated with insomnia, hypersomnia, sleep apnea, narcolepsy,nocturnal myoclonus, REM sleep interruptions, jet-lag, shift workers'sleep disturbances, dysomnias, night terror, night eating syndrome,insomnias associated with depression or with emotional/mood disorders,dysfunctions associated with sleep (parasomnias), as well as sleepwalking and enuresis, as well as sleep disorders which accompany aging.Sleep disorders and sleep disturbances are generally characterized bydifficulty in initiating or maintaining sleep or in obtaining restful orenough sleep.

In addition, certain drugs may also cause reductions in REM sleep as aside effect and the present invention may be used to correct those typesof sleeping disorders as well. The present invention would also be ofbenefit in the treatment of syndromes such as fibromyalgia which aremanifested by non-restorative sleep and muscle pain or sleep apnea whichis associated with respiratory disturbances during sleep. It will beclear to one skilled in the art that the present invention is notlimited to just sleep disorders and sleep disturbances, but isapplicable to a wide variety of conditions which result from adiminished quality of sleep.

The present invention is also concerned with treatment and prevention ofthese conditions, and with the use of a NPY Y5 antagonist, combinations,and compositions thereof, for the manufacture of a medicament useful fortreating or preventing these conditions.

In the present invention, it is preferred that the subject mammal is ahuman. Although the present invention is applicable both old and youngpeople, it may find greater application in elderly people. Further,although the invention may be employed to enhance the sleep of healthypeople, it may be especially beneficial for enhancing the sleep qualityof people suffering from sleep disorders or sleep disturbances.

The NPY Y5 antagonists of use in the present invention may be any NPY Y5antagonist known from the art.

The NPY Y5 antagonist may be peptidal or non-peptidal in nature,however, the use of a non-peptidal NPY Y5 antagonist is preferred. Inaddition, for convenience the use of an orally active NPY Y5 antagonistis preferred.

In the present invention, it is preferred that the NPY Y5 antagonistactive upon the central nervous system (CNS), such as the brain,following systemic administration, i.e. that it readily penetrates theCNS. Accordingly, a preferred NPY Y5 antagonist for use in the presentinvention is a CNS-penetrating NPY Y5 antagonist.

Non-limiting examples of Y5 receptor antagonists include compounds ofthe formula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of -Q-D;

D is selected from the group consisting of aryl or heteroaryl, whereinsaid aryl and heteroaryl groups may be optionally substituted, thesubstituent being selected from the group consisting of halogen, cyano,lower alkyl, halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, loweralkoxy, halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, loweralkanoyl and aryl;

n is an integer from 0 to 1;

Q is selected from the group consisting of a single bond or carbonyl;

T, U, V and W are each independently selected from the group consistingof nitrogen or a methylene group, said nitrogen or methylene group maybe optionally substituted with a substituent selected from the groupconsisting of: halogen, lower alkyl, hydroxy, and lower alkoxy;

X is selected from the group consisting of methylene or nitrogen;

Y is selected from the group consisting of nitrogen and oxygen, whereinsaid nitrogen may be optionally substituted with lower alkyl or oxygen;

and the pharmaceutically acceptable salts and esters thereof. Thesecompounds are further described and methods of preparing them can befound in International Publication Number WO 01/14376, and in U.S. Pat.Nos. 6,326,375, and 6,335,345, which are hereby incorporated byreference in their entirety.

Non-limiting examples of NPY Y5 receptor antagonists include compoundsof the formula:

or a pharmaceutically acceptable salt thereof, wherein;

V, W, X and Z are independently selected from CH and N;

R¹ is H, C₁₋₃ alkyl, C₁₋₃ alkoxy, F, or Cl;

R² is S(O)n R⁶, COR⁶ or CHO, wherein

n is 0, 1 or 2; and

R⁶ is N(R³)₂ or C₁₋₃ alkyl;

R³ is independently H or C₁₋₃ alkyl;

Ar is aryl or heteroaryl;

R⁴ and R⁵ are independently selected from:

-   -   (1) hydrogen,    -   (2) aryl, either unsubstituted or substituted with        -   (a) halo        -   (b) C₁₋₃ alkoxy,        -   (c) —N(C₁₋₃ alkyl)₂,        -   (d) C₂₋₄ alkanoyl, or        -   (e) aryl;    -   (3) nitro,    -   (4) C₁₋₅ alkyl,    -   (5) C₁₋₅ alkoxy,    -   (6) hydroxy-C₁₋₃ alkyl,    -   (7) carboxy,    -   (8) halo,    -   (9) C₁₋₅ alkylthio,    -   (10) C₁₋₅ ethoxycarbonyl,    -   (11) pyridylcarbonyl,    -   (12) benzoyl,    -   (13) phenyl-C₁₋₃ alkoxy,    -   (14) pyridyl, either unsubstituted or substituted with C₁₃ alkyl        or C₁₋₃ alkoxy,    -   (15) C₃₋₆ cycloalkyl,    -   (16) oxazolyl,    -   (17) thiazolyl,    -   (18) triazolyl,    -   (19) phenoxy, and    -   (20) C₂₋₆ alkanoyl.

These compounds are further described and methods of preparing them canbe found in International Publication Number WO 00/27845, and U.S. Pa.Nos. 6,191,160, and 6,313,298, which is hereby incorporated by referencein their entirety.

Non-limiting examples of NPY Y5 receptor antagonists include compoundL-152,804 of the formula:

and pharmaceutically acceptable salts, esters and tautomers thereof.Compound L-152,804 and its preparation are disclosed in J. OrganicChemistry, vol. 31, No. 5, p. 1639 (1966); and U.S. Pat. No. 6,258,837,which is hereby incorporated by reference in its entirety.

The above compounds are only illustrative of the NPY Y5 antagonistswhich are currently under investigation. As this listing of groups ofcompounds is not meant to be comprehensive, the methods of the presentinvention may employ any NPY Y5 antagonist and is not limited to anyparticular structural class of compound.

Suitable pharmaceutically acceptable salts of the NPY Y5 antagonists ofuse in the present invention include acid addition salts which may, forexample, be formed by mixing a solution of the compound with a solutionof a pharmaceutically acceptable non-toxic acid such as hydrochloricacid, fumaric acid, maleic acid, succinic acid, acetic acid, citricacid, tartaric acid, carbonic acid, phosphoric acid or sulphuric acid.Salts of amine groups may also comprise the quaternary ammonium salts inwhich the amino nitrogen atom carries an alkyl, alkenyl, alkynyl oraralkyl group. Where the compound carries an acidic group, for example acarboxylic acid group, the present invention also contemplates saltsthereof, preferably non-toxic pharmaceutically acceptable salts thereof,such as the sodium, potassium and calcium salts thereof.

Certain of the above defined terms may occur more than once in the aboveformula and upon such occurrence each term shall be definedindependently of the other. Similarly, the use of a particular variablewithin a noted structural formula is intended to be independent of theuse of such variable within a different structural formula.

Full descriptions of the preparation of the NPY Y5 antagonists which areemployed in the present invention may be found in the references citedherein.

The identification of a compound as a NPY Y5 antagonist, in particular aCNS penetrant NPY Y5 antagonist, and thus able to have utility in thepresent invention may be readily determined without undueexperimentation by methodology well known in the art, such as the assaysdescribed herein.

The NPY Y5 antagonist may be used alone or in conjunction with otheragents which are known to be beneficial in altering circadian rhythms orin the enhancement of sleep efficiency. The NPY Y5 antagonist and theother agent may be co-administered, either in concomitant therapy or ina fixed combination, or they may be administered at separate times. Forexample, the NPY Y5 antagonist may be administered in conjunction withother compounds which are known in the art to be useful for suppressingor stimulating melatonin production including melatonergic agents,noradrenergic and serotonergic re-uptake blockers, alpha-1-noradrenergicagonists, monamine oxidase inhibitors, other NPY agonists orantagonists; neurokinin-1 agonists; substance P; beta-adrenergicblockers and benzodiazepines, such as atenolol; or with other compoundswhich are known in the art to be useful for stimulating melatoninproduction including tricyclic antidepressants and alpha-2-adrenergicantagonists; or with melatonin precursors such as tryptophan,5-hydroxytryptophan, serotonin and N-acetylserotonin; as well asmelatonin analogs, melatonin agonists and melatonin antagonists, ormelatonin itself.

In addition, the NPY Y5 antagonist may be administered in conjunctionwith other compounds which are known in the art to be useful forenhancing sleep quality and preventing and treating sleep disorders andsleep disturbances, including e.g., sedatives, hypnotics, anxiolytics,antipsychotics, antianxiety agents, minor tranquilizers, melatoninagonists and antagonists, melatonin, melatonergic agents,benzodiazepines, barbiturates, 5HT-2 antagonists, and the like, such as:adinazolam, allobarbital, alonimid, alprazolam, amitriptyline,amobarbital, amoxapine, bentazepam, benzoctamine, brotizolam, bupropion,busprione, butabarbital, butalbital, capuride, carbocloral, chloralbetaine, chloral hydrate, chlordiazepoxide, clomipramine, cloperidone,clorazepate, clorethate, clozapine, cyprazepam, desipramine, dexclamol,diazepam, dichloralphenazone, divalproex, diphenhydramine, doxepin,estazolam, ethchlorvynol, etomidate, fenobam, flunitrazepam, flurazepam,fluvoxamine, fluoxetine, fosazepam, glutethimide, halazepam,hydroxyzine, imipramine, lithium, lorazepam, lormetazepam, maprotiline,mecloqualone, melatonin, mephobarbital, meprobamate, methaqualone,midaflur, midazolam, nefazodone, nisobamate, nitrazepam, nortriptyline,oxazepam, paraldehyde, paroxetine, pentobarbital, perlapine,perphenazine, phenelzine, phenobarbital, prazepam, promethazine,propofol, protriptyline, quazepam, reclazepam, roletamide, secobarbital,sertraline, suproclone, temazepam, thioridazine, tracazolate,tranylcypromaine, trazodone, triazolam, trepipam, tricetamide,triclofos, trifluoperazine, trimetozine, trimipramine, uldazepam,valproate, venlafaxine, zaleplon, zolazepam, zolpidem, and saltsthereof, and combinations thereof, and the like.

The NPY Y5 antagonist may be administered in conjunction with the use ofphysical methods such as with light therapy or electrical stimulation.In particular, the NPY Y5 antagonist may be administered in conjunctionwith scheduling bright light administration, ordinary-intensity lightexposure, or exposure to dim-light or darkness (or even sleep). In oneembodiment of the present invention, the NPY Y5 antagonist isadministered accompanied by having an individual wear dark or redgoggles at the time of administration to provide additive effects of thetreatment plus darkness. In another embodiment of the present invention,the individual wears dark goggles at times other than the time of NPY Y5antagonist administration to avoid the occurrence of an externalzeitgeber with respect to the phase shift resulting from the NPY Y5antagonist. Similarly, bright light exposure can be used in conjunctionwith administration of a NPY Y5 antagonist. As used herein, the term“light therapy” includes, but is not limited to, the above definitionsof light therapy.

Accordingly, the present invention further includes within its scope theuse of a NPY Y5 antagonist, alone or in combination with other agents,for altering circadian rhythms or for the prevention or treatment ofsleep disorders and sleep disturbances in a mammal. The preferred mammalfor purposes of this invention is human.

It will be appreciated to those skilled in the art that reference hereinto treatment extends to prophylaxis (prevention) as well as thetreatment of the noted diseases/disorders and symptoms.

Included within the scope of the present invention is the method ofusing a NPY Y5 antagonist for altering circadian rhythms or forenhancing and improving the quality of sleep. The NPY Y5 antagonist isuseful in enhancing or improving sleep quality as well as preventing andtreating sleep disorders and sleep disturbances in a mammal. Inaddition, the use of the NPY Y5 antagonist increases sleep efficiencyand augments sleep maintenance. The NPY Y5 antagonist may further beused in a method for preventing and treating sleep disorders and sleepdisturbances in a mammal. The present invention further provides apharmaceutical composition for altering circadian rhythms or forenhancing or improving sleep quality and increasing sleep efficiency andsleep maintenance.

The present method of using a NPY Y5 antagonist further provides thefollowing: an increase in the value which is calculated from the timethat a subject sleeps divided by the time that a subject is attemptingto sleep; a decrease in sleep latency (the time it takes to fallasleep); a decrease in the number of awakenings during sleep; a decreasein the time spent awake following the initial onset of sleep; anincrease in the total amount of sleep; an increase the amount andpercentage of REM sleep; an increase in the duration and occurrence ofREM sleep; a reduction in the fragmentation of REM sleep; an increase inthe amount and percentage of slow-wave (i.e. stage 3 or 4) sleep; anincrease in the amount and percentage of stage 2 sleep; a decrease inthe number of awakenings, especially in the early morning; an increasein daytime alertness; and increased sleep maintenance; enhancedcognitive function; and increased memory retention.

The present invention is further useful for the prevention and treatmentof sleep disorders and sleep disturbances including: sleep problemsassociated with insomnia, hypersomnia, sleep apnea, narcolepsy,nocturnal myoclonus, REM sleep interruptions, jet-lag, shift workers'sleep disturbances, dysomnias, night terror, insomnias associated withdepression or with emotional/mood disorders, as well as sleep walkingand enuresis, as well as sleep disorders which accompany aging,conditions associated with circadian rhythmicity, mental and physicaldisorders associated with travel across time zones and with rotatingshift-work schedules, or syndromes such as fibromyalgia which aremanifested by non-restorative sleep and muscle pain or sleep apnea whichis associated with respiratory disturbances during sleep.

In addition, the present invention includes within its scope apharmaceutical composition for enhancing and improving the quality ofsleep comprising, as an active ingredient, at least one NPY Y5antagonist in association with a pharmaceutical carrier or diluent. Thepresent invention further includes the use of a NPY Y5 antagonist in themanufacture of a medicament for achieving a circadian rhythmphase-shifting effect, alleviating a circadian rhythm disorder, blockingthe phase-shifting effects of light, enhancing and improving the qualityof sleep, or for the treatment of sleep disorders or sleep disturbances.

It will be known to those skilled in the art that there are numerouscompounds now being used to affect circadian rhythms or to enhance andimprove the quality of sleep. Combinations of these therapeutic agentssome of which have also been mentioned herein with a NPY Y5 antagonistwill bring additional, complementary, and often synergistic propertiesto enhance the desirable properties of these various therapeutic agents.In these combinations, the NPY Y5 antagonist and the therapeutic agentsmay be independently present in dose ranges from one one-hundredth toone times the dose levels which are effective when these compounds areused singly.

The NPY Y5 antagonist may be administered in combination with sedatives,hypnotics, anxiolytics, antipsychotics, antianxiety agents, minortranquilizers, melatonin agonists and antagonists, melatonergic agents,benzodiazepines, barbiturates, 5HT-2 antagonists, and the like, or theNPY Y5 antagonist may be administered in conjunction with the use ofphysical methods such as with light therapy or electrical stimulation.For example, to alter circadian rhythmicity or to enhance and improvethe quality of sleep a NPY Y5 antagonist may be given in combinationwith such compounds as: adinazolam, allobarbital, alonimid, alprazolam,amitriptyline, amobarbital, amoxapine, bentazepam, benzoctamine,brotizolam, bupropion, busprione, butabarbital, butalbital, capuride,carbocloral, chloral betaine, chloral hydrate, chlordiazepoxide,clomipramine, cloperidone, clorazepate, clorethate, clozapine,cyprazepam, desipramine, dexclamol, diazepam, dichloralphenazone,divalproex, diphenhydramine, doxepin, estazolam, ethchlorvynol,etomidate, fenobam, flunitrazepam, flurazepam, fluvoxamine, fluoxetine,fosazepam, glutethimide, halazepam, hydroxyzine, imipramine, lithium,lorazepam, lormetazepam, maprotiline, mecloqualone, melatonin,mephobarbital, meprobamate, methaqualone, midaflur, midazolam,nefazodone, nisobamate, nitrazepam, nortriptyline, oxazepam,paraldehyde, paroxetine, pentobarbital, perlapine, perphenazine,phenelzine, phenobarbital, prazepam, promethazine, propofol,protriptyline, quazepam, reclazepam, roletamide, secobarbital,sertraline, suproclone, temazepam, thioridazine, tracazolate,tranylcypromaine, trazodone, triazolam, trepipam, tricetamide,triclofos, trifluoperazine, trimetozine, trimipramine, uldazepam,valproate, venlafaxine, zaleplon, zolazepam, zolpidem, and saltsthereof, and combinations thereof, and the like, as well as admixturesand combinations thereof.

Typically, the individual daily dosages for these combinations may rangefrom about one-fifth of the minimally recommended clinical dosages tothe maximum recommended levels for the entities when they are givensingly.

To illustrate these combinations, a NPY Y5 antagonist effectiveclinically at a given daily dose range may be effectively combined, atlevels which are equal or less than the daily dose range, with thefollowing compounds at the indicated per day dose range: adinazolam,allobarbital, alonimid, alprazolam, amitriptyline, amobarbital,amoxapine, bentazepam, benzoctamine, brotizolam, bupropion, busprione,butabarbital, butalbital, capuride, carbocloral, chloral betaine,chloral hydrate, chlordiazepoxide, clomipramine, cloperidone,clorazepate, clorethate, clozapine, cyprazepam, desipramine, dexclamol,diazepam, dichloralphenazone, divalproex, diphenhydramine, doxepin,estazolam, ethchlorvynol, etomidate, fenobam, flunitrazepam, flurazepam,fluvoxamine, fluoxetine, fosazepam, glutethimide, halazepam,hydroxyzine, imipramine, lithium, lorazepam, lormetazepam, maprotiline,mecloqualone, melatonin, mephobarbital, meprobamate, methaqualone,midaflur, midazolam, nefazodone, nisobamate, nitrazepam, nortriptyline,oxazepam, paraldehyde, paroxetine, pentobarbital, perlapine,perphenazine, phenelzine, phenobarbital, prazepam, promethazine,propofol, protriptyline, quazepam, reclazepam, roletamide, secobarbital,sertraline, suproclone, temazepam, thioridazine, tracazolate,tranylcypromaine, trazodone, triazolam, trepipam, tricetamide,triclofos, trifluoperazine, trimetozine, trimipramine, uldazepam,venlafaxine, zaleplon, zolazepam, zolpidem, and salts thereof, andcombinations thereof, and the like, as well as admixtures andcombinations thereof. It will be readily apparent to one skilled in theart that the NPY Y5 antagonist may be employed with other agents toalter circadian rhythms or to control sleep disorders and sleepdisturbances in depressed patients and/or provide benefit in theprevention or treatment of sleep disorders and sleep disturbances.

Naturally, these dose ranges may be adjusted on a unit basis asnecessary to permit divided daily dosage and, as noted above, the dosewill vary depending on the nature and severity of the disease, weight ofpatient, special diets and other factors.

These combinations may be formulated into pharmaceutical compositions asknown in the art and as discussed below. A NPY Y5 antagonist may beadministered alone or in combination by oral, parenteral (e.g.,intramuscular, intraperitoneal, intravenous or subcutaneous injection,or implant), nasal, vaginal, rectal, sublingual, or topical routes ofadministration and can be formulated in dosage forms appropriate foreach route of administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert pharmaceutically acceptablecarrier such as sucrose, lactose, or starch. Such dosage forms can alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., lubricating agents such as magnesium stearate.Illustrative of the adjuvants which may be incorporated in tablets,capsules and the like are the following: a binder such as gumtragacanth, acacia, corn starch or gelatin; an excipient such asmicrocrystalline cellulose; a disintegrating agent such as corn starch,pregelatinized starch, alginic acid and the like; a lubricant such asmagnesium stearate; a sweetening agent such as sucrose, lactose orsaccharin; a flavoring agent such as peppermint, oil of wintergreen orcherry. In the case of capsules, tablets and pills, the dosage forms mayalso comprise buffering agents.

When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier such as fatty oil. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Tablets and pills can additionally beprepared with enteric coatings and tablets may be coated with shellac,sugar or both.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, the elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents. A syrup or elixir maycontain the active compound, sucrose as a sweetening agent, methyl andpropyl parabens as preservatives, a dye and a flavoring such as cherryor orange flavor.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Sterile compositions for injection may be formulatedaccording to conventional pharmaceutical practice by dissolving orsuspending the active substance in a vehicle such as water forinjection, a naturally occurring vegetable oil like sesame oil, coconutoil, peanut oil, cottonseed oil, etc., or a synthetic fatty vehicle likeethyl oleate or the like. Buffers, preservatives, antioxidants and thelike may be incorporated as required. Examples of non-aqueous solventsor vehicles are propylene glycol, polyethylene glycol, vegetable oils,such as olive oil and corn oil, gelatin, and injectable organic esterssuch as ethyl oleate. Such dosage forms may also contain adjuvants suchas preserving, wetting, emulsifying, and dispersing agents. They may besterilized by, for example, filtration through a bacteria-retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions. They canalso be manufactured in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. Compositions for rectal or vaginaladministration are preferably suppositories which may contain, inaddition to the active substance, excipients such as cocoa butter or asuppository wax. Compositions for nasal or sublingual administration arealso prepared with standard excipients well known in the art.

The dosage of active ingredient in the compositions of this inventionmay be varied, however, it is necessary that the amount of the activeingredient be such that a suitable dosage form is obtained. The activeingredient may be administered to patients (animals and human) in needof such treatment in dosages that will provide optimal pharmaceuticalefficacy. The selected dosage depends upon the desired therapeuticeffect, on the route of administration, and on the duration of thetreatment. As will be readily apparent to one skilled in the art, theeffect of a NPY Y5 antagonist which induces a phase shift in a centralcircadian pacemaker may be dependent on both the ambient and circadiantime of administration. The same compound may induce a phase advance, aphase delay or have minor effect on a particular circadian rhythmdepending on the circadian time of administration. The dose will varyfrom patient to patient depending upon the nature and severity ofdisease, the patient's weight, special diets then being followed by apatient, concurrent medication, the intrinsic NPY Y5 antagonist activityof the compound, the bioavailability upon oral administration of thecompound and other factors which those skilled in the art willrecognize.

In the treatment of a condition in accordance with the presentinvention, an appropriate dosage level, will generally be about 0.01 μgto 50 mg per kg patient body weight per day which may be administered insingle or multiple-doses. Preferably, the dosage level will be about 0.1μg to about 25 mg/kg per day; more preferably about 0.5 μg to about 10mg/kg per day. For example, for achieving a circadian rhythmphase-shifting effect, resetting the internal circadian clock,shortening the time of re-entrainment of circadian rhythms, alleviatinga circadian rhythm disorder, increasing alertness, or enhancing thequality of sleep, a suitable dosage level, is about 0.1 μg to 25 mg/kgper day, preferably about 0.5 μg to 10 mg/kg per day, and especiallyabout 1 μg to 5 mg/kg per day. In larger mammals, for example humans, atypical indicated dose is about 300 μg to 400 mg orally. A compound maybe administered on a regimen of several times per day, for example 1 to4 times per day, preferably once or twice per day. When using aninjectable formulation, a suitable dosage level is about 0.1 μg to 10mg/kg per day, preferably about 0.5 μg to 5 mg/kg per day, andespecially about 1 μg to 1 mg/kg per day. In larger mammals, for examplehumans, a typical indicated dose is about 100 μg to 100 mg i.v. Acompound may be administered on a regimen of several times per day, forexample 1 to 4 times per day, preferably once or twice per day.

Pharmaceutical compositions of the present invention may be provided ina solid dosage formulation preferably comprising about 100 μg to 500 mgactive ingredient, more preferably comprising about 100 μg to 250 mgactive ingredient. The pharmaceutical composition is preferably providedin a solid dosage formulation comprising about 100 μg, 1 mg, 5 mg, 10mg, 25 mg, 50 mg, 100 mg, 200 mg or 250 mg active ingredient.

The following examples are provided for the purpose of furtherillustration only and are not intended to be limitations on thedisclosed invention.

EXAMPLE 1

Y5 receptor antagonist assay:

To identify a potent Y5 antagonist for treatment of circadian rhythmdisorders in humans, the cloned human Y5 receptor is used in the primaryassay. Vectors expressing either the 455 amino acid form (See, e.g.,U.S. Pat. No 5,602,024) or a 10 amino acid, N-terminally shorter form(See, e.g., U.S. Pat. No. 5,919,901) can be introduced into cell linesto obtain cells which express the human Y5 receptor. Binding of[¹²⁵I]PYY (NEN) to membrane preparations from cells expressing thecloned human Y5 receptor are performed in 0.2 ml of 25 mM Tris buffer(pH 7.4) containing 10 mM MgCl₂, 1 mM PMSF, 0.1% bacitracin and 0.5%bovine serum albumin. Membranes (10-300 μg/ml) prepared from LMtk-,COS-7, HEK or CHO cells expressing Y5 receptors, are incubated at 25° C.for 120 min with [¹²⁵I]PYY (25 pM) in the presence of severalconcentrations of compounds to be evaluated. Bound and free peptides areseparated by filtration using a GF/C glass filter presoaked with 0.3%polyethylenimine. The remaining radioactivity on the filter isquantitated using a TopCount™ (Packard Instruments Co. Inc.). Specificbinding of [¹²⁵I]PYY is defined as the difference between total bindingand nonspecific binding in the presence of 1 μM PYY. The binding IC₅₀ iscalculated using GraphPad Prism (Ver. 3.0).

The functional potency of Y5 antagonists can be determined using variousassays which measure inhibition of second messenger pathways. NPYincreases intracellular Ca²⁺ concentration via activation of Y5receptors through coupling to Gαqi5. The potency of a Y5 antagonist inblocking NPY mediated Ca²⁺ increase can be used as a measure of itsfunctional antagonist activity. For example, CHO cells expressing bothNPY Y5 receptors and Gαqi5 are seeded (40,000 cells per well) into96-well plate 24 hr before assay. Cells are loaded for 1 hr with aCa²⁺-sensitive fluorescent dye, Fluo-4-AM in assay buffer (Hank'sBalanced Salts Solution (HBSS) containing 20 mM HEPES, 0.5 % BSA and 2.5mM probenecid, pH 7.4), washed 3 times with the assay buffer, thenreturned to the incubator for 1 hr before assay on a fluorometricimaging plate reader, FLIPR™ (Molecular Device, California). TheNPY-induced maximum change in fluorescence over baseline is determinedand the dose which induces a 50% increase in fluorescence is defined asthe EC₅₀ dose for NPY. To evaluate Y5 antagonists, the assay is repeatedwith the EC₅₀ dose of NPY in the presence of various concentrations of aY5 antagonist to generate a functional IC₅₀. The concentration-responsecurves are fitted using GraphPad Prism (Ver. 3.0). Using these assays,potent Y5 antagonists with a binding IC₅₀ and/or functional IC₅₀ of lessthan 1 μM can be identified. Useful antagonists would also have toposses other characteristics such as selectivity over the other NPYreceptors, good systemic exposure, sufficient half-life and brainpenetration.

EXAMPLE 2

Assessment of behavioral rhythms:

For the recording of locomotor activity rhythms in rodents, animalswould be maintained under a standard light-dark cycle (12:12 or 14:10,depending upon the species or strain of rodent) for several weeks priorto the start of each experiment. Animals would be housed individuallywith access to a running wheel in the cage and wheel-running activitywould be recorded continuously (e.g., using a Chronobiology kit,Actiview software, or another biological rhythm analysis softwarepackage). Food (rodent chow) and water would be available ad libitum.

(1) To evaluate whether NPY5 receptor antagonist treatment phase-shiftsthe circadian rhythm of locomotor activity, one would administer theNPY5 receptor antagonist or vehicle to animals at different circadiantimes while the animals were maintained under dim red light conditions(<15 lux of light) in “constant dark” conditions. Animals would be dosedwith the NPY5 receptor antagonist at times when one would expect toobserve phase delays (e.g., early subjective night, circadian time (CT)14) and phase advances (e.g., late subjective night, CT 19) inwheel-running activity to generate a phase-response curve.

(2) To evaluate the effects of NPY5 antagonists on light-induced phaseshifts of a rodent circadian locomotor activity rhythm, animals would behoused in constant darkness and then injected with the NPY5 antagonistor vehicle 30 minutes prior to exposure to a light-pulse at abehaviorally relevant time (i.e., at a time when light would producelarge phase advances or phase delays in behavior as outlined above; CT14and/or CT19). For light stimulation, the animals would be exposed to˜100-300 lux of fluorescent white light for 10-15 minutes. The locomotoractivity of the animals would be monitored for several days followingthe light pulse.

(3) To test whether injections of NPY5 antagonists mimic the effect ofdark pulses in animals housed in constant light, animals would be keptin constant light (˜100-300 lux). Dark pulses induce phase advances andphase delays in locomotor activity rhythms when applied, respectively,during the mid-subjective day and the late subjective night. A subset ofanimals would be treated with NPY5 antagonists or vehicle during themid-subjective day (e.g., CT6 or CT8) and another group of animals wouldbe similarly treated with NPY antagonists or vehicle during the latesubjective night (e.g., CT19).

(4) To evaluate whether daily NPY5 receptor antagonist treatment wouldentrain free-running locomotor activity rhythms in rodents maintained inconstant darkness (under dim red safelight;15 W, Kodak 1A filter),animals would be maintained in constant darkness for several days orweeks to stabilize their free-running activity rhythms. Animals would bedosed with an NPY5 receptor antagonist or vehicle for severalconsecutive days (e.g, ranging from 3-21 days) at the same clock time(e.g., CT10 or CT24). In this paradigm, the onset of wheel-runningactivity would “lock onto” the time at which the NPY5 receptorantagonist is administered. Entrainment would be defined as when theonset of locomotor activity coincided with the time of daily injections.

To determine phase-shifts in wheel-running rhythms for these studies,actograms (plots of activity data) would be examined and regressioncurves would be fitted by eye (or with the software) to the onsets oflocomotor activity for 7-10 days prior to the drug treatment andprojected to the day of treatment. This method would be used toextrapolate the magnitude of the phase-shift (i.e, the differencebetween the lines).

EXAMPLE 3

Determine the effect of a Y5 antagonist, L-152804 on phase shifts of thecircadian rhythm

A. Effect of L-152804 on circadian rhythms

Male golden hamsters are housed individually under a Light-Dark (LD)cycle of 14:10 upon arrival from the supplier for 2 weeks. All animalsare housed with a running wheel to measure activity. The animals arethen transferred to a constant dark (DD) cycle for 2 weeks and activityrecords from the running wheel are used to determine circadian time (CT)14.

Two groups of hamsters are treated orally with vehicle (0.5% methocel)or L-152804 at 50 mg/kg, then the animals are returned to their homecages and activity recorded for 10 days by monitoring the time theanimals spend on a running wheel. A shift in the peak time of activitywould indicate a Y5 antagonist can shift the circadian rhythm.

B. Effect of L-152804 on light induced phase shifts in the circadianrhythm

Male golden hamsters are housed individually under a Light-Dark (LD)cycle of 14:10 upon arrival from the supplier for 2 weeks. All animalsare housed with a running wheel to measure activity. The animals arethen transferred to a constant dark (DD) cycle for 2 weeks and activityrecords from the running wheel are used to determine circadian time (CT)14.

Two groups of hamsters are treated orally with vehicle (0.5% methocel)or L-152804 at 50 mg/kg, then subjected to light pulses at two differentbehaviorally-relevant times: CT14 and CT 20. Light pulses (503 nm,8.6×10¹² photons/cm2/sec, 5 min), which are known to induce a phaseshift in the activity level of hamsters of about ⅓ the maximal shift,are given to both groups. The animals are returned to their home cagesand activity recorded for 10 days. The Y5 antagonist treatment enhancesthe effect of the light pulse if the phase shift is greater then theeffect of vehicle treatment. Alternatively, the Y5 antagonist inhibitsthe light-induced phase shift if the phase shift is less than that seenwith the vehicle treated animals.

C. Effect of L-152804 activity induced phase shifts in the circadianrhythm

Male golden hamsters are housed individually under a Light-Dark (LD)cycle of 14:10 upon arrival from the supplier for 2 weeks. All animalsare housed with a running wheel to measure activity. The animals arethen transferred to a constant dark (DD) cycle for 2 weeks and activityrecords from the running wheel are used to determine circadian time (CT)8.

Two groups of hamsters are treated orally with vehicle (0.5% methocel)or L-152804 at 50 mg/kg, then subjected to a single injection of thebenzodiazepene agonist triazolam (3 mg/kg) at CT8. The animals arereturned to their home cages and activity recorded for 10 days todetermine if L-152804 suppresses the circadian shifts induced by theactivity stimulus, triazolam.

EXAMPLE 4

Double-Blind, Placebo-Controlled Study to Determine the Effect of aNeuropeptide Y Y5 antagonist on Light-Induced Melatonin Suppression inHealthy Young Men

The purpose of this study is to evaluate the effects of a neuropeptide YY5 antagonist on circadian rhythms in humans by examining the amount oflight-induced melatonin suppression in subjects treated with placebo orthe neuropeptide Y Y5 antagonist. If a neuropeptide Y Y5 antagonist isable to alter the amount of light-induced melatonin suppression and soinfluence circadian rhythms, it may be a useful agent, e.g., fortreating jet lag, shift workers, seasonal affective disorder, and sleepdisorders in the elderly.

This study is a double-blind, randomized, placebo-controlled, crossover,single-center study in healthy young men. After completing the screeningvisit, subjects follow a regular sleep/wake schedule for 2 weeks at homewhile wearing an actigraphy monitor in order to confirm theircompliance. After the 2-week period, subjects begin the in-laboratoryportion of the study, during which they will spend a baseline day inconstant routine (CR) conditions, a night of sleep in the laboratory,followed by another CR day and night, during which time subjects willnot go to sleep, but have a melatonin suppression test, followed by aday of recovery when subjects sleep and then are discharged beforenighttime. Two hours before their typical bedtime on Day 2, subjectsreceive orally either the neuropeptide Y Y5 antagonist L-152,804 orplacebo. Four hours later, 2 hours after their typical bedtime, subjectsare exposed to a 5-hour pulse of moderately bright light (300-900 lux).Following the light exposure, subjects stay in CR conditions for another5 hours, be allowed to sleep, and then leave the laboratory. Bloodsampling is performed on subjects throughout the in-laboratory visit inorder to collect samples for melatonin assays. Subjects remain at homefor a 3- to 8-week washout period before returning for the second partof the study. During the last 2 weeks before the subjects come back intothe laboratory, they follow a regular sleep/wake schedule for 2 weeks athome with actigraphic monitoring in order to confirm their compliance.Subjects then return to the laboratory to follow the same 3-day protocolbut they receive the opposite drug treatment (i.e. neuropeptide Y Y5antagonist or placebo). The primary response, suppression of melatonin,is assessed by calculating the percent change from baseline in themelatonin plasma during the 5-hour light pulse. The baseline value isdefined as the melatonin plasma AUC of the corresponding 5 hours whichoccurred 24 hours earlier.

The administration of an effective neuropeptide Y Y5 antagonist caninduce a change in the phase of the free-running circadian clock andblock the phase-shifting effects of light on the mammalian circadianclock.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various adaptations, changes, modifications,substitutions, deletions, or additions of procedures and protocols maybe made without departing from the spirit and scope of the invention.For example, effective dosages, other than the particular dosages as setforth herein above may be applicable as a consequence of variations inthe responsiveness of the mammal being treated for any of theindications with the compounds of the invention indicated above.Likewise, the specific pharmacological responses observed may varyaccording to and depending upon the particular active compounds selectedor whether there are present pharmaceutical carriers, as well as thetype of formulation and mode of administration employed, and suchexpected variations or differences in the results are contemplated inaccordance with the objects and practices of the present invention. Itis intended, therefore, that the invention be defined by the scope ofthe claims which follow and that such claims be interpreted as broadlyas is reasonable.

1. A method for achieving a circadian rhythm phase-shifting effect in amammal which comprises administering to the mammal an effective amountof a neuropeptide Y Y5 antagonist of structural formula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of-Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof:
 2. A method forresetting the internal circadian clock in a mammal which comprisesadministering to the mammal an appropriate amount of a neuropeptide Y Y5antagonist, or a pharmaceutically acceptable salt or ester thereof.
 3. Amethod for shortening the time of reentrainment of circadian rhythms ina mammal following a shift in the sleep-wake cycle which comprisesadministering to the mammal an appropriate amount of a neuropeptide Y Y5antagonist, or a pharmaceutically acceptable salt or ester thereof.
 4. Amethod for alleviating a circadian rhythm disorder in a mammal whichcomprises administering to the mammal an effective amount of aneuropeptide Y Y5 antagonist of structural formula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of -Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be- optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof.
 5. A method forthe treatment of a circadian rhythm disorder in a mammal which disorderis selected from the group consisting of: time-zone change (jet-lag)syndrome, shift-work sleep disorder, delayed sleep-phase syndrome,advanced sleep-phase syndrome, and non-24-hour sleep-wake disorder whichcomprises administering to the mammal an effective amount of aneuropeptide Y Y5 antagonist of structural formula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of -Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof.
 6. A method foralleviating the effects of jet lag in a mammal which comprisesadministering to the mammal an alertness increasing amount of aneuropeptide Y Y5 antagonist of structural formula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of -Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof.
 7. A method forenhancing the quality of sleep in a mammal which comprises administeringto the mammal an effective amount of a neuropeptide Y Y5 antagonist, ora pharmaceutically acceptable salt or ester thereof.
 8. The method ofclaim 1 wherein the mammal is a human.
 9. (canceled)
 10. (canceled) 11.(canceled)
 12. The method of claim 5 wherein the mammal is a human. 13.(canceled)
 14. The method of claim 1 wherein the neuropeptide Y Y5antagonist is administered in conjunction with melatonin or a compoundwhich suppresses or stimulates melatonin production
 15. The method ofclaim 1 wherein the neuropeptide Y Y5 antagonist is administered inconjunction with a compound which enhances sleep quality.
 16. The methodof claim 1 wherein the neuropeptide Y Y5 antagonist is administered inconjunction with light therapy.
 17. The method of claim 5 wherein theneuropeptide Y Y5 antagonist is administered in conjunction withmelatonin or a compound which suppresses or stimulates melatoninproduction.
 18. The method of claim 5 wherein the neuropeptide Y Y5antagonist is administered in conjunction a compound which enhancessleep quality.
 19. The method of claim 5 wherein the neuropeptide Y Y5antagonist is administered in conjunction with light therapy.
 20. Amethod for the prevention of a circadian rhythm disorder in a mammalwhich disorder is selected from the group consisting of: time-zonechange (jet-lag) syndrome, shift-work sleep disorder, delayedsleep-phase syndrome, advanced sleep-phase syndrome, and non-24-hoursleep-wake disorder which comprises administering to the mammal aneffective amount of a neuropeptide Y Y5 antagonist of structuralformula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of -Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof.
 21. A method foralleviating the effects of shift-work sleep disorder in a human in needthereof which comprises administering to the human an effective amountof a CNS-penetrating neuropeptide Y Y5 antagonist of structural formula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of -Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof.
 22. The method ofclaim 21 wherein the neuropeptide Y Y5 antagonist is administered inconjunction with melatonin or a compound which suppresses or stimulatesmelatonin production.
 23. The method of claim 21 wherein theneuropeptide Y Y5 antagonist is administered in conjunction with lighttherapy.
 24. A method for the treatment of a sleep disorder in a humanin need thereof which comprises administering to the human an effectiveamount of a CNS-penetrating neuropeptide Y Y5 antagonist of structuralformula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of -Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof.
 25. A method forthe prevention of a sleep disorder in a human in need thereof whichcomprises administering to the human an effective amount of aCNS-penetrating neuropeptide Y Y5 antagonist of structural formula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of -Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof.
 26. The method ofclaim 24 wherein the neuropeptide Y Y5 antagonist is administered inconjunction with melatonin or a compound which suppresses or stimulatesmelatonin production.
 27. The method of claim 24 wherein theneuropeptide Y Y5 antagonist is administered in conjunction with lighttherapy.
 28. A method for the treatment of a sleep disorder in anelderly human in need thereof which comprises administering to the humanan effective amount of a CNS-penetrating neuropeptide Y Y5 antagonist ofstructural formula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of-Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof.
 29. A method forthe prevention of a sleep disorder in an elderly human in need thereofwhich comprises administering to the human an effective amount of aCNS-penetrating neuropeptide Y Y5 antagonist of structural formula:

wherein A is selected from the group consisting of aryl or heteroaryl,wherein said aryl and heteroaryl groups may be optionally substituted oneither the carbon or hetero atom, the substituent being selected fromthe group consisting of halogen, nitro, lower alkyl, halo(lower)alkyl,hydroxy(lower)alkyl, cyclo(lower)alkyl, lower alkenyl, lower alkoxy,halo(lower)alkoxy, lower alkylthio, carboxyl, lower alkanoyl, loweralkoxycarbonyl, lower alkylene optionally substituted with oxo, and agroup represented by formula of -Q-D; D is selected from the groupconsisting of aryl or heteroaryl, wherein said aryl and heteroarylgroups may be optionally substituted, the substituent being selectedfrom the group consisting of halogen, cyano, lower alkyl,halo(lower)alkyl, hydroxy(lower)alkyl, hydroxy, lower alkoxy,halo(lower)alkoxy, lower alkylamino, di-lower alkylamino, lower alkanoyland aryl; n is an integer from 0 to 1; Q is selected from the groupconsisting of a single bond or carbonyl; T, U, V and W are eachindependently selected from the group consisting of nitrogen or amethylene group, said nitrogen or methylene group may be optionallysubstituted with a substituent selected from the group consisting of:halogen, lower alkyl, hydroxy, and lower alkoxy; X is selected from thegroup consisting of methylene or nitrogen; Y is selected from the groupconsisting of nitrogen and oxygen, wherein said nitrogen may beoptionally substituted with lower alkyl or oxygen; and thepharmaceutically acceptable salts and esters thereof.
 30. The method ofclaim 28 wherein the neuropeptide Y Y5 antagonist is administered inconjunction with melatonin or a compound which suppresses or stimulatesmelatonin production.
 31. The method of claim 28 wherein theneuropeptide Y Y5 antagonist is administered in conjunction with lighttherapy. 32-36. (canceled)